Banana streak virus (BSV) is causing increasing concern in almost every producing area of banana and plantain (Musa spp.) worldwide. This situation appeared partially linked to some breeding lines and micropropagated hybrids. A complete BSV sequence integrated into the genome of a triploid plantain has been recently characterised and it has been hypothesised that it could give rise to infectious virus via recombination. In this study, we evaluated the effect of a routine micropropagation procedure on the expression of BSV in the FHIA 21 tetraploid hybrid. The widespread presence of integrated sequences and the absence of episomal BSV in thirty FHIA 21 "mother plants" selected for micropropagation were first confirmed by specific PCR and IC-PCR tests. The proliferation stage of the procedure, characterised by an intensive production of neoformed buds, appeared determinant in BSV expression whereas the rooting and acclimatisation stages had little or no effect. The duration in culture and the way of subdividing the clumps of proliferation influenced greatly the percentage of episomal BSV infections, reaching 58% of infected micropropagated lines after six in vitro subcultures. These data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.

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